PROJECT SUMMARY The worldwide epidemic of type 2 diabetes is associated with early and continued functional pancreatic beta cell loss and cardiovascular disease (CVD) due to accelerated atherosclerosis. Our data suggest that the signal transducer and activator of transcription 4 (STAT4) functions as a central mediator of inflammation in the beta cell and in the aorta. We recently demonstrated that global deletion of STAT4 prevents high fat diet induced adipose inflammation and atherosclerosis. We provide the first evidence that STAT4 is expressed in neutrophils and IL-12-induced STAT4 and p66Shc activation plays a role in neutrophil activation and functions. STAT4 role in myeloid cells has not been well-established and nothing is yet known about the role of STAT4 in neutrophils. We also report that STAT4 regulates macrophage functions in atherosclerosis, but it is not known how STAT4 and its downstream targets regulate macrophage functions. Our overall hypothesis is that the IL- 12/STAT4 axis is one of the key signals for macrophages and neutrophils that shape inflammatory functions of these cells, at least in part, by p66Shc-dependent mechanisms leading to atherosclerosis, beta cell functional decline and glucose intolerance. We propose three Specific Aims to address this hypothesis. Specific Aim 1 will address a question how STAT4 impacts neutrophil functions, identify upstream signaling events resulting in STAT4 activation in neutrophils with a specific focus on the IL-12-induced Jak1/JNK/p66Shc-dependent pathway. We will also test a neutrophil-specific role of STAT4 in atherosclerosis using diabetogenic diet with added cholesterol (DDC) fed atherosclerosis-prone Ldlr-/- mice that have a targeted deletion of STAT4 in neutrophils. Specific Aim 2 will investigate myeloid cell-specific role of STAT4 in the regulation of glucose intolerance and islet inflammation upon atherosclerosis. We will use myeloid cell-specific STAT4 deficient DDC fed Ldlr-/- mice and examine mechanisms by which STAT4 affects intra-islet immune composition, islet inflammation, and beta cell health and functions. We will also evaluate key upstream and downstream pathways regulating STAT4-induced beta cell dysfunction using siRNA-p66Shc, and quantitative sensitive proteomics. To separate the impact of macrophages vs neutrophils in the induction of islet dysfunction, we will use neutrophil-specific Stat4-/-Ldlr-/- mice for some experiments in this aim. Aim 3 will examine to what extent IL-12/STAT4 signaling in myeloid cells supports inflammasome activation, aortic M? retention, defective efferocytosis, and accelerated atherogenesis in Ldlr-/- mice. We will test a role of the JAK1-JNK-p38-p66Shc- STAT4 pathway in these processes. Overall, the results from this project will alter existing paradigms, as it presents an original hypothesis about a novel STAT4-dependent common pathway that via targeting M?s and N?s affects progression of atherogenesis and islet dysfunction. The completed studies could lead to new therapeutic advances to reduce tissue and cellular immune inflammation and thus maintain insulin sensitivity, beta cell function and reduce atherosclerosis.